In our organism, many nerve cells form a network that enables high-order sensory functions and body regulation. Neurons extend nerve fibres, which transmit information between cells by means of electrical signals. This network is established during development by the extension of nerve fibres, and by adulthood the network is largely established. In adulthood, when the peripheral nerves are damaged, they can regenerate by extending fibres again. The central nervous system, on the other hand, does not regenerate once damaged. Therefore, once the central nervous system is damaged in a spinal cord injury, there is a possibility of residual damage.

In experiments using rats, it has been reported that central nerve fibres regenerate when cAMP is administered (Fig. 1). We therefore focused on PC12D cells, which elongate projections induced by cAMP, because we thought that clarifying the molecular mechanism by which cAMP induces projection elongation would provide fundamental knowledge for the regeneration of the central nervous system.

Figure 1. horizontal section of rat spinal cord. Nerve fibres in orange. Normally do not regenerate 8 weeks after injury, but do regenerate when cAMP is administered (*),Neuman et al, Neuron 2002.

Known downstream of cAMP are PKA (Protein Kinase A), a serine-threonine phosphatase, and Epac, a cAMP-GEF. Knockout experiments showed that PKA is important for neurite elongation. On the other hand, small G proteins such as Rac1 and Cdc42 are important for cytoskeletal organisation such as neurite elongation. In other words, investigating how PKA regulates Rac1 and Cdc42 will provide a molecular mechanism for cAMP-induced neurite elongation. For this purpose, we used FRET, a technique that allows real-time measurement of molecular activity in living cells (Fig. 2).

Figure 2. intracellular FRET observation; the FRET biosensor changes its structure in response to molecular activity and FRET occurs internally; by detecting changes in the biosensor's fluorescence properties due to FRET, molecular activity can be quantified.

FRET imaging of the activity of Rac1, Cdc42, PIP3 and PKA during cAMP-induced neurite outgrowth showed a common pattern of activity for PKA and Rac1: immediately after cAMP administration, PKA and Rac1 are activated throughout the cell, but then their activity is localised to the neurite (Fig. 3). The activity of PKA and Rac1 was also found to be localised to the neurite (Fig. 3). This suggested that PKA activated by cAMP may localise to the neurite and directly regulate Rac1. We investigated the relationship between PKA and STEF (Tiam2), a Rac1-specific GEF, and found that PKA phosphorylates the 749th threonine of STEF, increasing GEF activity and activating Rac1. These results are one of the few reports of PKA directly regulating Rac1 and provide new insights into cAMP-mediated neural regeneration.

Fig. 3. real-time measurement of Rac1 and PKA during cAMP-induced neurite outgrowth FRET images of PC12D cells expressing Raichu-Rac1 (top) for Rac1 activity and AKAR (bottom) for PKA activity, respectively. Warm colors indicate higher activity; time course after cAMP administration. Cell-wide activity is shown up to 30 min after administration, after which the activity is localized to the protrusions during the process of neurite outgrowth.